(FOR RESEARCH USE ONLY. DO NOT USE IT IN CLINICAL DIAGNOSIS!) cgmp ELISA Kit (Direct Competitive) Based on Monoclonal Anti-cGMP Antibody Catalog No: E-EL-DS02 96T This manual must be read attentively and completely before using this product. If you have any problems, please contact our Technical Service Center for help. Phone: 240-252-7368(USA) 240-252-7376(USA) Email: techsupport@elabscience.com Website: Please kindly provide us the lot number (on the outside of the box) of the kit for more efficient service. Copyright 2017-2018 Elabscience Biotechnology Inc. All Rights Reserved
Introduction Guanosine 3, 5 -cyclic monophosphate (cgmp) is a key second messenger molecule in intracellular signal transduction. Monitoring cgmp levels is one of the most common ways to screen for agonists and antagonists of G protein coupled receptors (GPCRs). Elabscience cgmp ELISA Kit uses Competitive-ELISA as the method. The microtiter plate provided in this kit has been pre-coated with goat anti-mouse IgG. During the reaction, sample or standard, cgmp- HRP Conjugate and mouse monoclonal anti-cgmp antibody (cgmp MAb) are sequentially added to each wells. cgmp in the sample or standard competes with a fixed amount of HRP-labeled cgmp for the binding sites of cgmp MAb. Free components are washed from the plate. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of cgmp in the samples is then determined by comparing the OD of the samples to the standard curve. The ELISA typically displays an IC 50 (50% B/B 0) of approximately 5 pmol/ml and a detection limit of approximately 0.1 pmol/ml. Kit components Item Specification Storage Goat anti-mouse IgG Micro-plate (12 8 wells) 96 wells 4 cgmp Standard (10000 pmol/ml) 0.1 ml -80 cgmp-hrp Conjugate (1000 ) 0.02 ml -80 Anti-cGMP McAb (1000 ) 0.02 ml -80 Assay Buffer (10 ) 30 ml 4 Neutralizing Buffer 8 ml 4 Sample Diluent 30 ml 4 TMB Substrate 18 ml 4 Stop Solution 8 ml 4 Other supplies required Microplate reader with 450 nm wavelength filter High-precision transferpettor, EP tubes and disposable pipette tips 37 Incubator Deionized Absorbent paper Loading slot for Wash Buffer Eppendorf tubes 2
Sample treatment 1. Tissue samples: Tissue samples should be frozen in liquid nitrogen. Then weigh the frozen tissue and add 5-10 volume of 0.1 M HCl. Homogenize the sample on ice using a homogenizer. Centrifuge at > 600 g at room temperature for 5 min. The supernatant may be assayed directly or be diluted with the 0.1 M HCl. (If the concentration of camp in samples exceed the detection range of kit) 2. Cells samples: After removing the culture medium, add moderate amount of 0.1 M HCl to lyse cells (adding 0.1% to 1% triton x-100 to the 0.1 M HCl can enhance the lysis). Incubate for 10 min and visually inspect the cells to verify cell lysis. If the cells are not be lysed adequately, incubate for a further 10 min. Centrifuge at 600 g at room temperature for 5 min, then collect the supernatant and take the assay directly. When used in this concentration range, the detergent will not interfere with the binding portion of the assay, however there will be a modest increase in the optical density. Samples containing triton should be evaluated against a standard curve diluted in the same for the most accurate determination. The supernatants can then be used directly in the assay. 3. Urine, Serum and Culture Medium Samples: After treating 1 ml of the supernatant media with 10 µl of concentrated hydrochloric acid, Centrifuge urine or plasma for 5 min at 600 g at room temperature. The supernatants can then be used directly in the assay or diluted to a suitable multiple. Both phosphodiesterases and immune globulin can also be removed by 5% TCA precipitation or by using 10 KD molecular weight cut off micro centrifuge filters. 3
Reagent preparation 1. cgmp standard solution Label eight tubes 1# through 8#. Pipet 990 μl 0.1 M HCl into tube 1# and 500 μl 0.1 M HCl into tubes 2-8#. Add 10 μl of the 10000 pmol/ml standard to tube 1#. Vortex thoroughly. Add 500 μl of tube 1# to tube 2# and vortex thoroughly. Continue this for tubes 3# through 8#. The concentration of cgmp in tube 1# through 8# will be 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 pmol/ml respectively. Diluted standards should be used within 15 min of preparation. Label one tube as the Zero Standard (0 pmol/ml, maximum binding/b 0). Pipet 500 μl 0.1 M HCl into this tube. 1. Assay Buffer Prepare the assay buffer by diluting 30 ml of the supplied Assay Buffer (10 ) with 270 ml of deionized water. This can be stored at room temperature until the kit expiration date, or for 3 months, whichever is earlier. 2. cgmp-hrp conjugate Dilute camp-hrp Conjugate (1000 ) to 1 camp-hrp conjugate working solution with assay buffer before use. 3. Anti-cGMP McAb Dilute Anti-cAMP McAb (1000 ) to 1 Anti-cAMP McAb working solution with assay buffer before use. 4
Assay procedure All standards (including maximum binding (B 0)) and samples should be added the same volume. Neutralizing Buffer and vortex for 2 seconds immediately after the addition. 1. Pipet 50 µl of the Neutralizing Buffer into each well, except the TA (Total Activity) and blank wells. 2. Pipet 50 µl of 0.1 M HCl into the NSB (Non-Specific Binding) and the maximum binding (B 0) wells. 3. Pipet 50 µl of Standards into the appropriate wells. 4. Pipet 50 µl of the Samples into the appropriate wells. 5. Pipet 50 µl of assay buffer into the NSB wells. 6. Pipet 50 µl of cgmp-hrp Conjugate into each well except the TA and blank wells. 7. Pipet 50 µl of Anti-cGMP McAb into each well, except the Blank, TA and NSB wells. 8. Incubate the plate at room temperature for 2 hours. 9. Empty the wells and rinse four times with assay buffer, remove any remaining solution. 10. Add 5 µl of the Conjugate to the TA wells. 11. Add 150 µl of the Substrate solution to every well. Incubate at room temperature for about 10 min without shaking. 12. Add 50 µl of Stop Solution to every well. This stops the reaction and the plate should be read immediately. 13. Monitor the absorbance increase at 450 nm using an absorbance plate reader, preferably with correction at 630 nm. If the plate reader is not able to be blanked against the Blank wells, manually subtract the mean optical density of the Blank wells from all readings. 5
Well Neutralizing 0.1M Standard cgmp - HRP Anti-cGMP Assay Buffer HCl /Sample Conjugate McAb Buffer Blank (μl) - - - - - - TA (μl) - - - 5 - - NSB (μl) 50 50-50 - 50 B 0 (μl) 50 50-50 50 - Standard /sample (μl) 50-50 50 50-1 2 3 4 5 6 7 8 9 10 11 12 A Blank S1 S1 SP1 SP16 SP17 SP32 SP33 SP48 SP49 SP64 SP65 B Blank S2 S2 SP2 SP15 SP18 SP31 SP34 SP47 SP50 SP63 SP66 C NSB S3 S3 SP3 SP14 SP19 SP30 SP35 SP46 SP51 SP62 SP67 D NSB S4 S4 SP4 SP13 SP20 SP29 SP36 SP45 SP52 SP61 SP68 E B0 S5 S5 SP5 SP12 SP21 SP28 SP37 SP44 SP53 SP60 SP69 F B0 S6 S6 SP6 SP11 SP22 SP27 SP38 SP43 SP54 SP59 SP70 G B0 S7 S7 SP7 SP10 SP23 SP26 SP39 SP42 SP55 SP58 SP71 H TA S8 S8 SP8 SP9 SP24 SP25 SP40 SP41 SP56 SP57 SP72 Blank- contains substrate only. TA-Total activity, contains HRP conjugate (5 µl) and substrate. NSB-Non-specific binding, contains neutralizing buffer, HRP conjugate and substrate. B 0: 0 pg/ml standard, contains neutralizing buffer, HRP conjugate, antibody and substrate. S1-S8: Standards 1-8 SP1-SP72: samples. Notes for operation: 1. Bring all kit reagents to warm to room temperature(20-25 ) before use. 2. Pipet standards and samples to the bottom of the wells. 3. Add the reagents to the side of the well to avoid contamination. 4. This kit uses break-apart microtiter strips, which allow the user to measure as many samples as desired. Unused wells must be kept desiccated at 4 in the sealed bag provided. The wells should be used in the frame provided. 5. Prior to addition of substrate, ensure that there is no residual wash buffer in the wells. 6. Stop solution is caustic, be careful during operation. 6
Calculation 1. Calculate the average Net Optical Density (OD) bound for each standard and sample by subtracting the average NSB OD from the average OD bound: Average Net OD = Average Bound OD - Average NSB OD 2. Using Logit-Log paper plot Average Net OD or Percent Bound (B/Bo) versus concentration of camp for the standards. The concentration of camp in the unknowns can be determined by interpolation. B/Bo= Average Net OD Average Net Bo OD 3. The lower limit of detection was determined as the concentration corresponding to a signal two standard deviations above the mean of the zero standard. The LOD is ~0.1 pmol/ml. Typical standard curve A new standard curve must be generated for each assay performed. 7
Cross reactivity The cross reactivity for a number of related compounds were determined by competition ELISA assays. Potential cross reactants were dissolved in the kit Assay Buffer at concentrations from 500,000 to 500 pmol/ml. These samples were then measured in the cgmp assay, and the measured cgmp concentration at 50% B/B 0 calculated. The % cross reactivity was calculated by comparison with the actual concentration of cross reactant in the sample and expressed as a percentage. Compound Cross Reactivity cgmp 100% GMP <0.0001% GTP <0.0001% cgmp <0.0001% AMP <0.0001% ATP <0.0001% cump <0.0001% CTP <0.0001% Copyright 2017-2018 Elabscience Biotechnology Inc. All Rights Reserved 8